Treatment of cardiac arrhythmias

ABSTRACT

Compositions of the invention, including compounds that bind to a receptor for a glucagon-like peptide-1, an incretin, a glucagon-like peptide-1 (GLP-1), an exendin, or an agonist, an analog (preferably an agonist analog), a derivative, or a variant of any of aforementioned compounds, are used in the prevention and treatment of arrhythmias associated with cardiac ischemia, cardiac ischemia-perfusion and/or congestive heart failure. The invention relates to both the method and compositions for such treatment.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/783,917, filed Mar. 4, 2013, which is a divisional of U.S.application Ser. No. 12/750,575, filed Mar. 30, 2010, issued as U.S.Pat. No. 8,389,473, which is a divisional of U.S. application Ser. No.10/740,146, filed Dec. 17, 2003, issued as U.S. Pat. No. 7,790,681,which claims the benefit of priority to U.S. Provisional Application No.60/434,508, filed Dec. 17, 2002, and U.S. Provisional Application No.60/434,888, filed Dec. 19, 2002, each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to compositions and methods for preventingcardiac arrhythmias using a compound that binds to a receptor for aglucagon-like peptide-1, an incretin, a glucagon-like peptide-1 (GLP-1),an exendin, or an agonist, an analog (preferably an agonist analog), aderivative, or a variant of any of aforementioned compounds andfragments thereof.

BACKGROUND OF THE INVENTION

Cardiac arrhythmias and ischemic heart disease afflict an estimated 20million Americans, and possibly ten times as many people worldwide. Ifleft undetected and untreated, they often result in heart attacks anddeaths.

An arrhythmia is an irregular heartbeat. The heart beats on its own dueto its natural pacemaker, a small cluster of specialized cells calledthe sinoatrial node (S-A node). The S-A node is located in the rightatrium and produces electrical signals at regular intervals that aresent through a pathway in the heart muscle. The S-A node signals followa natural electrical pathway that helps the heart beat efficiently. Anelectrical impulse travels from the S-A node through theatrioventricular node (A-V node), a second cluster of cells located nearthe center of the heart. The A-V node then sends the signals out to thewalls of the ventricles.

Normally, the two ventricles contract a fraction of a second after theyhave been filled with blood from an atrial contraction. This timingsequence is called atrio-ventricular synchrony. Sometimes, however,something goes wrong with the heart's electrical system, and theheartbeat becomes arrhythmic. An arrhythmia can occur when: (1) the S-Anode develops an abnormal rate or rhythm; (2) the normal electricalpathway is interrupted, or (3) another part of the heart tries to takeover as the pacemaker. Though there are several types of arrhythmias,they all have the commonality of preventing the heart from pumping bloodefficiently.

Fast, abnormal heart rhythms, usually over 100 beats per minute, arecalled tachyarrhythmias. When the heart's electrical signals come fromthe ventricle instead of the S-A node, this causes an arrhythmia knownas ventricular tachycardia (VT). As the heart beats faster, it pumpsless blood because there is not enough time for the heart to fill withblood between beats. If this fast heartbeat continues, the brain andbody may not receive enough blood and oxygen, causing fainting spells,blackouts, temporary blind spots or dizziness. Eventually, the patientmay become unconscious and in extreme cases the heart may stop (cardiacarrest). The most common cause of arrhythmias is heart disease,particularly coronary artery disease, abnormal heart valve function, andheart failure.

VT is a frequent precursor to another type of arrhythmia, ventricularfibrillation (VF). In VF, the heart beats much faster than normal,sometimes over 300 beats a minute. The ventricles “quiver” during VF anddo not carry out coordinated contractions. Because little blood ispumped from the heart, VF is a form of cardiac arrest and is fatalunless treated immediately.

Arrhythmias complicate all forms of cardiac disease. Ventriculartachycardia and fibrillation occur commonly in the setting of ischemicheart disease and congestive heart failure (CHF). In the setting ofmyocardial infarction, ventricular arrhythmias may develop secondarilyto ischemia or reperfusion. Reperfusion occurs subsequent to therapiesthat reestablish flow in an artery that is obstructed by a blood clot,i.e. thrombolytic agents or following an intervention, such asangioplasty, coronary bypass grafting or placement of an intracoronarystent.

A major problem in congestive heart failure is stress hyperglycemia andinsulin resistance. As a result of the combination of high circulatinglevels of free fatty acids and reduced glucose uptake, there is a shifttoward fatty acid oxidation, depletion of Krebs cycle intermediates anddiminished glucose oxidation. These changes ultimately lead to reducedlevels of CrP and loss of energy reserve.

Although the mechanisms underlying ventricular arrhythmias are complexand not fully understood, it has been established that glycolysis playsan important role as the source of ATP to maintain the electrochemicalgradient across the cardiac cellular membrane. Potassium (K⁺), calcium(Ca²⁺), and sodium (Na⁺) gradients are all modulated by ATP that arisesfrom glycolysis. Moreover, inhibition of glycolysis is arrhythmogenic,while glucose-insulin-potassium (GIK) infusions in the setting ofischemia are anti-arrhythmic.

Conventional treatment for arrhythmias is aimed at decreasing pacemakeractivity and modifying impaired conduction. These treatments usuallyinvolve the use of sodium channel blockers, calcium channel blockersand/or beta blockers in an effort to decrease the automaticity,conduction, and excitability of the heart or increase the refractoryperiod of cardiac muscle. While drug treatments are often effectiveagainst arrhythmias, drugs frequently have side effects and require thepatient to remember to take them on a daily basis. Mild to moderate sideeffects associated with these drugs include drowsiness, dizziness,nausea, bradycardia, and low blood pressure, while more severe sideeffects include torsades des pointes (a form of VT) and even suddendeath. Further, these drugs can actually cause arrhythmias at increaseddosages due to their toxic effects on cardiac conduction at theselevels.

Artificial pacemakers are also frequently used in the treatment ofarrhythmias. Pacemakers are electronic devices that act in place of theheart's own pacemaker and are programmed to imitate the normalconduction sequence of the heart. Usually they are implanted surgicallybeneath the skin of the chest and have wires running to the heart. Thereare several disadvantages associated with the use of pacemakers,including the need to replace the units every 8-10 years and theirpotential to be interfered with by certain types of equipment, such asmagnetic resonance imaging machines (MRIs).

Therapy for arrhythmias can also include devices that deliver a shock tothe heart to stop an abnormal rhythm and restore a normal one. Using anelectric shock for this purpose is called cardioversion, electroversion,or defibrillation. Usually in this procedure, a large machine thatdelivers a shock (a defibrillator) is used by a team of doctors andnurses to stop a life-threatening arrhythmia. More recently, adefibrillator about the size of a pack of cards can be implantedsurgically in the patient. These small devices, which automaticallysense life-threatening arrhythmias and deliver a shock, are used inpeople who would otherwise die when their heart suddenly stops. Becausethese defibrillators do not prevent arrhythmias, the patient usuallymust also take drugs as well.

There is, therefore, a need in the art for a safe and effectivecomposition for preventing and treating cardiac arrhythmias. It is aprimary objective of this the present invention to fulfill this need.

SUMMARY OF THE INVENTION

The invention describes compositions and methods for reducing the riskof suffering from, preventing, or treating cardiac arrhythmias.Compositions of the invention include a compound that binds to areceptor for a glucagon-like peptide-1, an incretin, a glucagon-likepeptide-1 (GLP-1), an exendin, or an agonist, an analog (preferably anagonist analog), a derivative, or a variant of any of them, as well asbiologically fragments thereof.

The present inventors have recognized that compositions of theinvention, including GLP-1 and exendins, have anti-arrhythmic effects inpatients with cardiac ischemia, cardiac ischemia-reperfusion, andcongestive heart failure. For example, GLP-1 has been found to reducecardiac injury and enhance recovery in patients with these disorders.Incretins, including GLP-1, are glucose-dependent insulinotropichormones. GLP-1 and exendin effectively enhance peripheral glucoseuptake without inducing dangerous hypoglycemia. They also stronglysuppress glucagon secretion, independent of its insulinotropic action,and thereby powerfully reduce plasma free fatty acid (FFA) levelssubstantially more than can be accomplished with insulin. High FFAlevels have been implicated as a major toxic mechanism during myocardialischemia.

Accordingly, it is a primary objective of the present invention toprovide compositions and methods for preventing and treating cardiacarrhythmias.

It is a further objective of the present invention to providecompositions and methods for preventing and treating cardiac arrhythmiasthat are effective in patients having cardiac ischemia, cardiacischemia-reperfusion, and congestive heart failure.

It is yet a further objective of the present invention to providecompositions and methods for preventing and treating cardiac arrhythmiasthat reliably reduce injury associated with reperfusion and ischemia,and enhance patient recovery.

It is a further objective of the present invention to providecompositions and methods for preventing and treating cardiac arrhythmiaswithout the side effects and disadvantages of conventional therapies.

Moreover, the compounds of the invention may be administered by anyconventional means, including subcutaneously, intravenously, orally,transmucosally, intraperitoneally, or other means known in the art. Thecompositions are particularly useful in treating arrhythmias resultingfrom ischemic heart disease and congestive heart failure.

Thus, in one aspect, the invention contemplates a method for preventingand treating arrhythmias comprising administering to an individual aneffective amount of a composition which includes a compound which bindsto a receptor for glucagon-like peptide-1, an incretin, a glucagon-likepeptide-1 (GLP-1), an exendin, or an agonist, an analog (preferably anagonist analog), a derivative, or a variant of any of aforementionedcompounds, and biologically active fragments thereof.

In one embodiment, methods of the invention include administeringcompositions of the invention at a dose from about 0.1 pmol/kg/min. upto about 10 pmol/kg/min. Other dose ranges may be from about 0.01pmol/kg to 20 nmol/kg. Further contemplated are a single or multipleinjection(s) in a dose from about 0.005 nmol/kg to 20 nmol/kg.

In another embodiment, methods of the invention include a concurrentadministration with any one or more of a glucose, a potassium, a freeradical scavenger or an anti-oxidant.

In yet other embodiments of the invention, the compositions of theinvention are administered within four hours of an ischemic event andmay be continued following the ischemic event. The composition may beadministered concurrently or as soon as possible following therapiesthat reestablish flow in an artery that has been obstructed, such asangioplasty, coronary bypass grafting, and placement of an intracoronarystent.

The compositions of the invention may be administered to treatventricular arrhythmias. The ventricular arrhythmia may be caused by acondition selected from the group consisting of cardiac ischemia,cardiac ischemia-reperfusion, and congestive heart failure.

In another general aspect, methods of the invention include metabolicintervention with a composition that includes a compound which binds toa receptor for glucagon-like peptide-1, an incretin, a glucagon-likepeptide-1 (GLP-1), an exendin, or an agonist, an analog (preferably anagonist analog), a derivative, or a variant of any of aforementionedcompounds, and fragments thereof to prevent or treat cardiacarrhythmias, said method comprising administering to an individual inneed of such treatment an effective amount of a composition whichincludes a compound which binds to a receptor for glucagon-likepeptide-1, an incretin, a glucagon-like peptide-1 (GLP-1), an exendin,or an agonist, an analog (preferably an agonist analog), a derivative,or a variant of any of aforementioned compounds, and biologically activefragments thereof.

The method and means of accomplishing each of the above objectives aswell as others will become apparent from the detailed description of theinvention, which follows hereafter.

DETAILED DESCRIPTION

The present invention relates to the development of compositions for theprevention and treatment of cardiac arrhythmias using an incretin, aglucagon-like peptide-1 (GLP-1), an exendin, a compound that binds to areceptor for glucagon-like peptide-1, or an agonist, an analog(preferably an agonist analog), a derivative, or a variant of any of theaforementioned compounds and biologically active fragments thereof. Onepremise forming the basis of this discovery is that compounds of theinvention, including GLP-1, are effective at maintaining theelectrochemical gradient across cardiac cellular membranes, therebyreducing the likelihood of arrhythmias developing.

Cardiac arrhythmias can develop due to a variety of factors. Forinstance, arrhythmias may develop secondary to ischemia or reperfusion.Heart muscle is largely dependent on uninterrupted blood flow, whichguarantees delivery of oxygen and substrates to cells while washing outharmful metabolic products. Ischemia, e.g. resulting from decrease orcessation of myocardial blood flow, leads to rapid changes in myocardialmetabolism. The degree of these changes is highly dependent upon theseverity of the ischemia. For anatomical and physiological reasons,contractile myocytes in endocardium are the most vulnerable cells.Ischemia is a dynamic process. With rapid reperfusion, full recovery ofmyocardial metabolism occurs; but continuation of ischemia leads tototal tissue necrosis in a few hours. Reperfusion, although generallyconsidered beneficial, can cause tissue injury by several mechanisms,including oxidative stress, and thus affect the final recovery of thecontractibility.

Total cessation of myocardial blood flow leads to rapid perturbations inmyocardial metabolism. In a few seconds, oxygen dissolved in cytoplasmor bound to myoglobin is consumed, seriously disturbing oxidativephosphorylation and mitochondrial ATP production. Levels of high energyphosphates, mainly creatine phosphate and ATP, are decreased, and thebreakdown products of adenine nucleotides, such as inorganic phosphateand adenosine, accumulate.

Liberation of free fatty acids in lipolysis is stimulated in myocardialischemia by increased circulating catecholamines, but fatty acidoxidation and tricarboxylic acid cycle are inhibited. This leads tocytosolic accumulation of free fatty acid CoA-esters and inhibition ofadenine nucleotide translocase. Glycogenolysis and anaerobic glycolysisare stimulated, leading to accumulation of lactate and H⁺-ions andintracellular acidosis. Finally, the accumulation of proteins, lactate,and reduced form of NADH leads to inhibition of glycolysis and anaerobicenergy production through glycolysis. The energy-dependent transmembranecontrol is lost, with intracellular K⁺ and Mg²⁺ ions leaking out of thecells and extracellular Na⁺ and Ca²⁺ ions entering the cells. Theredistribution of electrolytes leads to osmotic changes and cellularedema.

It is believed that several of the electrolytic changes that occurduring ischemia may be responsible for cardiac arrhythmias. First, asnoted above, during ischemia, intracellular Na⁺ increases. Duringreperfusion, this results in depolarization and short action potentialscombined with low extracellular K⁺. Dispersion is pronounced andfavorable to arrhythmias.

Second, systolic and mitochondrial Ca²⁺ levels increase during ischemiaand reperfusion. An increase in cystolic Ca²⁺ activates a number ofchannels, carriers, and enzymes and modulates others, which results indelayed after depolarizations and arrhythmias.

Further, during ischemia, amphiphiles and fatty acids accumulate in theplasma membrane, the gap junction, and the intracellular membranes ofthe SR and the mitochondria. Amphiphiles and fatty acids may interactdirectly with channel proteins, with the phospholipids surrounding thechannel proteins, or changing the membrane fluidity. Amphiphilesincrease inward current at the resting potential with simultaneousreduction of outward current through K⁺ channels. Fatty acids activateoutward currents and stimulate the K⁺/Ca²⁺ exchanger. The simultaneousactivation of inward and outward currents favors K⁺ loss and Ca²⁺overload, creating conditions that generate arrhythmias. (Cameliet,1999).

It is also believed that reperfusion injury may manifest clinically asreperfusion arrhythmias. Early reperfusion is an absolute prerequisitefor the survival of ischemic tissue. Although ultimately necessary forrecovery, reperfusion is often considered a double-edged sword, and canactually lead to worsening of tissue injury by various mechanisms. Aswith ischemia, reperfusion is associated with Ca²⁺ overload throughactivation of the K⁺/Ca²⁺ exchanger, thereby creating conditionsfavorable to cardiac arrhythmias.

Complications associated with congestive heart failure include stresshyperglycemia and insulin resistance. As a result of the combination ofhigh circulating levels of free fatty acids and reduced glucose uptake,there is a shift toward fatty acid oxidation. Again, these fatty acidscan activate outward currents through K⁺ channels, and stimulate theK⁺/Ca²⁺ exchanger. The simultaneous activation of inward and outwardcurrents favors K⁺ loss and Ca²⁺ overload, thereby creating conditionsfavorable to the generation of arrhythmias.

GLP-1 and exendin are glucose-dependent insulinotropic peptides thateffectively enhance peripheral glucose uptake without inducing dangeroushypoglycemia. Further, they strongly suppress glucagon secretion,independent of their insulinotropic action, and thereby powerfullyreduce plasma free fatty acid (FFA) levels substantially more potentlythan can be accomplished with insulin (i.e., greater FFA suppression atequivalent prevailing insulin concentrations that are submaximallyeffective).

The present inventors have now discovered that GLP-1, exendins, andother compositions of the invention can be effective in the preventionand treatment of cardiac arrhythmias. It has now been found that thedual capacity of GLP-1 to powerfully stimulate insulin release andreduce insulin resistance provides this molecule with the unique abilityto prevent and treat cardiac arrhythmias by enhancing glucose uptake andmetabolism, at the expense of reduced FAA metabolism, into cardiacmuscle. In this respect, incretins, GLP-1, exendins, compounds that binda GLP-1 receptor, and agonists, analogs, derivatives, and variantsthereof, as well as their active fragments can be especially effectivein preventing and treating arrhythmias in patients with cardiacischemia, cardiac ischemia-reperfusion, and/or congestive heart failure.

Treatment with GLP-1 and other compositions of the invention may enhanceglycolysis in patients and shift the balance from fatty acid towardsglucose oxidation. These effects prevent loss of potassium and calciumoverload, and reduce the risk of cardiac arrhythmias.

Compositions of the invention may also stimulate the secretion ofendogenous insulin and therefore can be used to achieve all of thebeneficial actions attributed to an insulin infusion in the metabolictreatment of arrhythmias. Although high-dose GIK infusions typicallycontain 25-33% glucose and 50-100 U insulin/L, the requirement forintroduction of hyperglycemia per se to achieve therapeutic efficacy,versus only providing a metabolic milieu for the safe administration ofhigh doses of insulin, is unclear. It is likely that adequate bloodglucose levels are required to enable substrate delivery, but this doesnot necessarily imply a need for hyperglycemia and should not detractfrom the fact that insulin exerts important effects other than glucoseuptake. Therefore, a therapeutic infusion of a composition of theinvention, including GLP-1 and exendin, may require a modest (e.g., 5%)glucose administration in order to maintain blood glucose at slightlyabove physiological levels in order to trigger insulin release. Glucoseis not required as a safety measure, since blood levels of ≦3.5 mMabrogate the insulin-stimulating activity of GLP-1 and exendin, therebycompletely protecting against the dangers of hypoglycemia.

Insulin resistance (IR) has been recognized increasingly as a majorpathogenic factor for multiple systemic diseases, and not only inindividuals having Type-2 diabetes. Although many patients with Type-2diabetes manifest insulin resistance, many individuals with IR do nothave diabetes. An important recent insight has been the realization thatIR is an independent risk factor for the development and severity ofcardiovascular diseases, including ischemia-reperfusion injury and leftventricle dysfunction. IR is strongly associated with severe heartdisease, both acutely and chronically, which leads to the enhanced andpotentially damaging use by the heart of fatty acids as a fuel source inpreference to glucose. Administration of GLP-1, exendin and othercompositions of the invention, can reverse the use of fatty acids asfuel to glucose, thereby reducing free fatty acids and preventing thedevelopment of conditions favorable to the development of cardiacarrhythmias. The administration of GLP-1, exendin and other compositionsof the invention may be especially effective in the treatment ofventricular arrhythmias.

The administration of GLP-1, exendin, and other compositions of theinvention, should be effective in a majority of patients withoutrequiring concurrent glucose administration. However, a small proportionof subjects may require glucose to elicit an adequate insulin response.In addition, it also may be necessary to administer potassium to correctexcess shifts of potassium in the intracellular compartment when glucoseis co-administered with compositions of the invention.

In addition to the use of GLP-1, exendin and other compositions of theinvention, the methods of the invention can include use of free radicalscavengers or anti-oxidants such as glutathione, melatonin, Vitamin E,and superoxide dismutase (SOD). In such combinations, reperfusion damagerisk can be lessened even further.

Compositions of the invention include a compound that binds to areceptor for a glucagon like peptide-1, an incretin, a glucagon-likepeptide-1 (GLP-1), an exendin, or an agonist, an analog (preferably anagonist analog), a derivative, or a variant of any of the aforementionedcompounds, as well as biologically active fragments thereof. An“agonist” includes any compound that mimics at least one of the actionsof an incretin, a GLP-1, or an exendin, as described herein.

An “analog” includes any peptide whose sequence was derived from that ofthe base receptor-binding compound, incretin, GLP-1, or exendin, whetheror not including insertions, substitutions, extensions, or deletions,preferably having at least 50 or 55% amino acid sequence identity withthe base molecule, more preferably having at least 70%, 80%, 90%, or 95%amino acid sequence identity with the base molecule. Such analogs maycomprise conservative or non-conservative amino acid substitutions(including non-natural amino acids or as well as D forms), and if it isan “agonist analog,” exhibits at least one characteristic of the basemolecule, preferably having a potency better than the base molecule, orwithin five orders of magnitude of the base molecule, more preferably 4,3, 2, or 1 order of magnitude when evaluated by art-known measures.

A “derivative” includes any base molecule or analog having a chemicalmodification within, attached or linked to, or associated with themolecule. Such chemical modifications can include internal linkers(e.g., spacing or structure-inducing) or appended molecules, such asmolecular weight-enhancing molecules (e.g., polyethylene glycol (PEG)),or tissue targeting molecules. Examples of such molecules are known inthe art, for example, insulinotropic peptides, including GLP-1 andexendin, modified with a maleimide group are described in U.S. Pat. No.6,593,295, incorporated herein by reference.

A “variant” includes any modification to the base molecule, analog orvariant not encompassed in the terms “analog” and “derivative,” as wouldbe known to a person of ordinary skill in the art. For example, variantsmay include proforms or chimeras of a selected molecule. Small moleculesare included in the compounds useful in the invention to the extent thatthey bind to a receptor for GLP-1 or exendin. Not all of the peptidemolecules described as incretins, glucagon-like peptide-1 (GLP-1),exendins, or analogs, derivatives, or variants may bind to a receptorfor GLP-1, although they are still useful in the invention by virtue ofa pharmacology not dependent on a known GLP-1 receptor. These moleculesmay still possess the desired biological activities described herein.Other compounds encompassed within the scope of the invention includethose described in U.S. Pat. Nos. 6,569,832; 6,528,486; 6,514,500;6,458,924; 6,451,987; 6,451,974; 6,268,343, all herein incorporated byreference.

An example of a base molecule, as the term is used above, is GLP-1, alsoknown as glucagon-like peptide-1 [7-36] amide (also referred to as GLP-1[7-36]NH₂), a product of the proglucagon gene having the amino acidsequence His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu GlyGln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg-NH₂ (SEQ IDNO:1). It is secreted into plasma mainly from the gut and produces avariety of biological effects related to pancreatic and gastrointestinalfunction.

Many functions of GLP-1[7-36]NH₂, “GLP-1,” as used herein, are known(e.g., Orskov, et al., Diabetes, 42:658-61, 1993; D'Alessio, et al., J.Clin. Invest., 97:133-38, 1996, Williams B, et al., J Clin EndocrinolMetab 81 (1): 327-32, 1996; Wettergren A, et al., Dig Dis Sci 38 (4):665-73, 1993; Schjoldager B T, et al., Dig Dis Sci 34 (5): 703-8, 1989;O'Halloran D J, et al., J Endocrinol 126 (1): 169-73, 1990; WettergrenA, et al., Dig Dis Sci 38 (4): 665-73, 1993). GLP-1[7-37], which has anadditional glycine residue at its carboxy terminus, also stimulatesinsulin secretion in humans (Orskov, et al., Diabetes, 42:658-61, 1993).

Compositions of the invention include GLP-1 agonist analogs. By “agonistanalog” is meant a compound that mimics at least one effect of GLP-1 asdescribed above. This definition of agonist analog could includecompounds that bind to a receptor or receptors where GLP-1 causes theparticular effect. Certain GLP-1 analogs with agonist activity aredescribed in Chen et al., U.S. Pat. No. 5,512,549, issued Apr. 30, 1996,entitled Glucagon-Like Insulinotropic Peptide Analogs, Compositions andMethods of Use. Other GLP-1 analogs with agonist activity are describedin Johnson et al., U.S. Pat. No. 5,574,008, issued Nov. 12, 1996,entitled, Biologically Active Fragments of Glucagon-Like InsulinotropicPeptide. Still other GLP-1 analogs with agonist activity are describedin Buckley et al., U.S. Pat. No. 5,545,618, issued Aug. 13, 1996,entitled GLP-1 Analogs Useful for Diabetes Treatment. All threereferenced U.S. patents are incorporated herein by this reference. Thepresent invention includes the use of recombinant human GLP-1 analogsand GLP-1 analogs derived from other species, whether recombinant orsynthetic.

In certain aspects, the GLP-1 agonist analogs used in the methods of thepresent invention can be GLP-1(7-34) and GLP-1(7-35), as disclosed inU.S. Pat. No. 5,118,666, herein incorporated by reference, as well asGLP-1(7-37) as disclosed in U.S. Pat. No. 5,120,712, herein incorporatedby reference. Also included are GLP-1 analogs having a reduced tendencyto aggregate such as those described in WO 01/98331; GLP-1 analogs thathave N-terminal truncation, U.S. Pat. No. 5,574,008; GLP-1 analogs withattached acyl groups, U.S. Pat. No. 5,512,549; and GLP-1 analogs thatare amidated, WO 02/48192; and GLP-1 analogs of U.S. patent applicationSer. No. 10/276,772, all of which are incorporated by reference.

Additional analogs include, GLP-1 analogs at position 8, U.S. Pat. No.5,981,488, incorporated by reference. In brief, analogs include those offormula (XI),R₁-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R₂(SEQ ID NO:33) or a pharmaceutically acceptable salt thereof, wherein:

R₁ is selected from the group consisting of His, D-histidine,desamino-histidine, 2-amino-histidine, .beta.-hydroxy-histidine,homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine;X is selected from the group consisting of Met, Asp, Lys, Thr, Leu, Asn,Gln, Phe, Val, and TyrY and Z are independently selected from the group consisting of Glu,Gln, Ala, Thr, Ser, and Gly, and;R₂ is selected from the group consisting of NH₂, and Gly-OH; providedthat, if R₁ is His, X is Val, Y is Glu, and Z is Glu, then R₂ is NH₂.

V8-GLP-1 and other position 8 analogs can be found in U.S. Pat. No.5,705,483, incorporated by reference. In brief, analogs include those offormula (XII),R₁-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R₂(SEQ ID NO: 34) wherein:

R₁ is selected from the group consisting of L-histidine, D-histidine,desamino-histidine, 2-amino-histidine, .beta.-hydroxy-histidine,homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine;X is selected from the group consisting of Ala, Gly, Val, Thr, Ile, andalpha-methyl-Ala;Y is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, andGly;Z is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, andGly;R₂ is selected from the group consisting of NH₂, and Gly-OH; providingthat the compound has an isoelectric point in the range from about 6.0to about 9.0 and further providing that when R₁ is His, X is Ala, Y isGlu, and Z is Glu, R₂ must be NH₂.

In other aspects, the GLP-1 agonist analogs are variants or analogs ofGLP-1 known in the art, such as, for example, Gln⁹-GLP-1(7-37),D-Gln⁹-GLP-1(7-37), acetyl-Lys⁹-GLP-1(7-37), Thr¹⁶-Lys¹⁸-GLP-1(7-37),and Lys¹⁸-GLP-1(7-37). Derivatives of GLP-1 are also contemplated in thepresent invention and include, for example, acid addition salts,carboxylate salts, lower alkyl esters, and amides (see, e.g.,WO91/11457). Generally, the various forms of GLP-1 are known tostimulate insulin secretion (insulinotropic action) and cAMP formation(see, e.g., Mojsov, S., Int. J. Peptide Protein Research, 40:333-343(1992)).

In still other aspects, the present invention contemplates GLP-1agonists of the general formula (I):

wherein R₁ is selected from the group consisting of4-imidazopropionyl(des-amino-histidyl), 4-imidazoacetyl, or4-imidazo-alpha, alpha dimethyl-acetyl;

R₂ is selected from the group consisting of C₆-C₁₀ unbranched acyl, oris absent;

R₃ is selected from the group consisting of Gly-OH or NH₂; and,

Xaa₄₀ is Lys or Arg.

In one embodiment, the GLP-1 agonists are naturally-occurringGLP-1(7-37) that arise from adding various R groups via a peptide bondto the amino terminus of the peptide portion of Formula I (SEQ ID NO:2).Optionally, further compounds of the invention are made by acylating theepsilon amino group of the Lys34 residue and by making limited aminoacid substitutions at position 26 or by altering the carboxy terminus.

It should be noted that for the above formula, the nomenclature schemeused is that which has been developed around processed forms of GLP-1.In this scheme, the amino terminus of the known GLP-1(7-37) OH has beenassigned number 7 and the carboxy terminus number 37. Therefore, thefirst Ala residue of Formula I corresponds to residue 8 ofGLP-1(7-37)OH. Likewise Xaa₄₀ in Formula I corresponds to residue 26 ofGLP-1(7-37)OH, and so forth.

In still other aspects, the present invention providesbiologically-active GLP-1 fragments of formula (II):

(SEQ ID NO: 3) R₄-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Xaa₄₁-Gly-Arg-R₅

wherein R₄ is selected from the group consisting of:

a) H₂ N; b) H₂ N-Ser; c) H₂ N-Val-Ser; d) H₂ N-Asp-Val-Ser;(SEQ ID NO: 4) e) H₂ N-Ser-Asp-Val-Ser; (SEQ ID NO: 5)f) H₂ N-Thr-Ser-Asp-Val-Ser; (SEQ ID NO: 6)g) H₂ N-Phe-Thr-Ser-Asp-Val-Ser; (SEQ ID NO: 7)h) H₂ N-Thr-Phe-Thr-Ser-Asp-Val-Ser; (SEQ ID NO: 8)i) H₂ N-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser; (SEQ ID NO: 9)j) H₂ N-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser; or (SEQ ID NO: 10)k) H₂ N-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser;

Xaa₄₁ is selected from the group consisting of Lys or Arg; and

wherein R₅ is selected from the group consisting of NH₂, OH, Gly-NH₂, orGly-OH.

In still other aspects, the invention provides modified forms of theGLP-1(7-34); (7-35); (7-36) or (7-37) human peptide or the C-terminalamidated forms thereof. The native peptides have the amino acid sequence(SEQ ID NO:11):

7     10        15        20        25H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-   30                37I-A-W-L-V-K-(G)-(R)-(G)wherein (G), (R), and (G) are present or absent depending on theindicated chain length. The modified forms contain one or morealterations of the native structure and are of improved ability fortherapeutic use. Either the modified forms have greater potency thanglucagon to potentiate insulin secretion or enhanced stability in plasmaor both.

The analogs of the invention which show enhanced insulin stimulatingproperties may have the foregoing sequence, or a C-terminal amidethereof, with at least one modification of SEQ ID NO:11, selected fromthe group consisting of:

(a) substitution of a neutral amino acid, arginine, or a D form oflysine for lysine at position 26 and/or 34 and/or a neutral amino acid,lysine, or a D form of arginine for arginine at position 36;(b) substitution of an oxidation-resistant amino acid for tryptophan atposition 31;(c) substitution according to at least one of:

Y for V at position 16;

K for S at position 18;

D for E at position 21;

S for G at position 22;

R for Q at position 23;

R for A at position 24; and

Q for K at position 26;

(d) a substitution comprising at least one of:

an alternative small neutral amino acid for A at position 8;

an alternative acidic amino acid or neutral amino acid for E at position9;

an alternative neutral amino acid for G at position 10; and

an alternative acidic amino acid for D at position 15; and

(e) substitution of an alternative neutral amino acid or the D orN-acylated or alkylated form of histidine for histidine at position 7.

With respect to modifications (a), (b), (d) and (e), the substitutedamino acids may be in the D form, as indicated by a superscript †, e.g.,C^(†). The amino acids substituted at position 7 can also be in theN-acylated or N-alkylated forms.

In another aspect, the invention is directed to peptides which showenhanced degradation resistance in plasma as compared to GLP-1(7-37)wherein this enhanced resistance to degradation is defined as set forthbelow. In these analogs, any of the above-mentioned truncated forms ofGLP-1(7-34) to GLP-1(7-37) or their C-terminal amidated form is modifiedby

(a) substitution of a D-neutral or D-acidic amino acid for H at position7, or(b) substitution of a D-amino acid for A at position 8, or(c) both, or(d) substitution of an N-acylated or N-alkylated form of any naturallyoccurring amino acid for H at position 7.

Thus, analogs of the invention which are resistant to degradationinclude (N-acyl (1-6C) AA)⁷ GLP-1(7-37) and (N-alkyl (1-6C) AA)⁷GLP-1(7-37) wherein when AA is a lysyl residue, one or both nitrogensmay be alkylated or acylated. AA symbolizes any amino acid consistentwith retention of insulin stimulating activity.

For substitutions of D-amino acids in the 7 and 8 positions of SEQ IDNO:11, the D residue of any acidic or neutral amino acid can be used atposition 7 and of any amino acid at position 8, again consistent withinsulin stimulating activity. Either or both of position 7 and 8 can besubstituted by a D-amino acid; the D-amino acid at position 7 can alsobe acylated or alkylated as set forth above. These modified forms areapplicable not only to GLP-1(7-37) but also the shorter truncatedanalogs as set forth above.

Other modified GLP-1s, as well as exendins, useful in the practice ofthe claimed invention can be found in U.S. Pat. No. 6,528,486, which isincorporated by reference.

As previously stated, GLP-1 analogs, as well as exendin analogs, may bepeptides containing one or more amino acid substitutions, additions,extensions, or deletions, compared with GLP-1(7-36)amide, exendin-4 orexendin-3. In one embodiment, the number of substitutions, deletions, oradditions is 30 amino acids or less, 25 amino acids or less, 20 aminoacids or less, 15 amino acids or less, 10 amino acids or less, 5 aminoacids or less or any integer in between these amounts. In one aspect ofthe invention, the substitutions include one or more conservativesubstitutions. A “conservative” substitution denotes the replacement ofan amino acid residue by another, biologically active similar residue asis well known in the art. Examples of conservative substitutions includethe substitution of one hydrophobic residue, such as isoleucine, valine,leucine, or methionine for another, or the substitution of one polarresidue for another, such as the substitution of arginine for lysine,glutamic for aspartic acids, or glutamine for asparagine, and the like.

It is further understood that GLP-1 analogs include the above describedpeptides which have been chemically derivatized or altered, for example,peptides with non-natural amino acid residues (e.g., taurine, β- andγ-amino acid residues and D-amino acid residues), C-terminal functionalgroup modifications, such as amides, esters, and C-terminal ketonemodifications and N-terminal functional group modifications, such asacylated amines, Schiff bases, or cyclization, as found, for example, inthe amino acid pyroglutamic acid. Exendin analogs may have similarmodifications.

Also included in the present invention are peptide sequences havinggreater than 50% or 55% amino acid sequence identity, and preferablygreater than 70, 80, 90, or 95% amino acid sequence identity to SEQ IDNOs:1, 12, and 14, as well as truncated sequences thereof. As usedherein, sequence identity refers to a comparison made between twomolecules using standard algorithms well known in the art. The preferredalgorithm for calculating sequence identity for the present invention isthe Smith-Waterman algorithm, for example, SEQ ID NO: 1 [i.e.,GLP-1(1-37)], SEQ ID NO:12 or 14 [exendin-3 and 4, respectively] can beused as the reference sequences to define the percentage identity ofhomology over their length. The choice of parameter values for matches,mismatches, and insertions or deletions is arbitrary, although someparameter values have been found to yield more biologically realisticresults than others. One preferred set of parameter values for theSmith-Waterman algorithm is set forth in the “maximum similaritysegments” approach, which uses values of 1 for a matched residue and −⅓for a mismatched residue (a residue being either a single nucleotide orsingle amino acid). Waterman, Bull. Math. Biol. 46; 473 (1984).Insertions and deletions (indels), x, are weighted as x_(k)=1+⅓k, wherek is the number of residues in a given insert or deletion. Id.

For instance, a sequence that is identical to the 37-amino acid residuesequence of SEQ ID NO: 1, except for 18 amino acid substitutions and aninsertion of 3 amino acids, would have a percent identity given by:

[(1×37 matches)−(⅓×18 mismatches)−(1+3/3 indels)]/37=78% “identity.”

This algorithm can be used with any amino acid sequence to determinesequence homology.

Agonists of glucagon-like peptide that exhibit activity through aGLP-1(7-36)amide receptor have been described. See EP 0708179 A2; Hjorthet al., J. Biol. Chem. 269; 30121 (1994); Siegel et al., Amer. DiabetesAssoc. 57^(th) Scientific Session, Boston (1997); Hareter et al., Amer.Diabetes Assoc. 57^(th) Scientific Session, Boston (1997); Adelhorst etal., J. Biol. Chem. 269, 6275 (1994); Deacon et al., 16^(th)International Diabetes Federation Congress Abstracts, DiabetologiaSupplement (1997); Irwin et al., Proc. Natl. Acad. Sci. USA 94; 7915(1997); Mojsov, Int. J. Peptide Protein Res. 40; 333 (1992). Göke &Byrne, Diabetic Medicine 13; 854 (1996). Recent publications discloseBlack Widow GLP-1 and Ser² GLP-1. See Holz & Hakner, Comp. Biochem.Physiol., Part B 121; 177 (1998) and Ritzel et al., J. Endocrinol 159;93 (1998).

GLP-1 receptors are cell-surface proteins found, for example, oninsulin-producing pancreatic β-cells; the GLP-1(7-36) receptor has beencharacterised in the art. Additional receptors at which GLP-1 andexendins act are also thought to exist, and may mediate effects by whichthe instant invention is operative. Methods of determining whether achemical or peptide binds to or activates a particular GLP-1 receptorare known to the skilled artisan. For example, U.S. Pat. Nos. 6,051,689,5,846,747, and 5,670,360 describe GLP-1 receptors, as well as methodsfor using them. The contents of the patents are incorporated byreference.

The biological activity of a GLP-1 agonist and/or analog can bedetermined by in vitro and in vivo animal models and human studies, asis well known to the skilled artisan. GLP-1 biological activity can bedetermined by standard methods, in general, by receptor binding activityscreening procedures, which involve providing appropriate cells thatexpress the GLP-1 receptor on their surface, for example, insulinomacell lines such as RINmSF cells or INS-1 cells. See Mojsov, Int. J.Peptide Protein Res. 40; 333 (1992) and EP 0708179 A2. Cells that areengineered to express a GLP-1 receptor also can be used. In addition tomeasuring specific binding of tracer to membrane using radioimmunoassaymethods, cAMP activity or glucose dependent insulin production can alsobe measured. In one method, a polynucleotide encoding a GLP-1 receptoris employed to transfect cells so that they express the GLP-1 receptorprotein. Thus, for example, these methods may be employed for screeningfor a receptor agonist by contacting such cells with compounds to bescreened and determining whether such compounds generate a signal (i.e.,activate the receptor). Other screening techniques include the use ofcells that express the GLP-1 receptor, for example, transfected CHOcells, in a system to measure extracellular pH or ionic changes causedby receptor activation. For example, potential agonists may be contactedwith a cell that expresses the GLP-1 protein receptor and a secondmessenger response (e.g., signal transduction or ionic or pH changes),may be measured to determine whether the potential agonist is effective.

Polyclonal and monoclonal antibodies can be utilized to detect, purify,and identify GLP-1-like peptides for use in the methods describedherein. Antibodies such as ABGA1178 detect intact GLP-1(1-37) orN-terminally-truncated GLP-1(7-37) or GLP-1(7-36)amide. Other antibodiesdetect the end of the C-terminus of the precursor molecule, a procedurethat allows one—by subtraction—to calculate the amount of biologicallyactive, truncated peptide (i.e., GLP-1(7-37)amide). Orskov et al.,Diabetes 42; 658 (1993); Orskov et al., J. Clin. Invest. 1991, 87; 415(1991).

GLP-1, its agonists, analogs, derivatives, variants, and biologicallyactive fragments, that are peptides can be made by solid-state chemicalpeptide synthesis. Such peptides can also be made by conventionalrecombinant techniques using standard procedures described in, forexample, Sambrook & Maniatis, Molecular Cloning, A Laboratory Manual.“Recombinant,” as used herein, means that a gene is derived from arecombinant (e.g., microbial or mammalian) expression system that hasbeen genetically modified to contain a polynucleotide encoding a GLP-1peptide as described herein.

GLP-1, its agonists, analogs, derivatives, variants, and biologicallyactive fragments, that are peptides may be a naturally purified product,or a product of synthetic chemical procedures, or produced byrecombinant techniques from prokaryotic or eukaryotic hosts (forexample, by bacteria, yeast, higher plant, insect, or mammalian cells inculture or in vivo). Depending on the host employed in a recombinantproduction procedure, the polypeptides of the present invention aregenerally non-glycosylated, but may be glycosylated. The GLP-1 peptidescan be recovered and purified from recombinant cell cultures by methodsincluding, but not limited to, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography, and lectinchromatography. High-performance liquid chromatography (HPLC) can beemployed for final purification steps.

Other compositions of the invention include exendins, which refer tonaturally occurring exendin peptides that are found in Gila-monster.Preferred exendins include exendin-3 (SEQ ID NO:12), which is present inthe salivary secretions of Heloderma harridum, exendin-4 (SEQ ID NO:14),which is a peptide present in the salivary secretions of Helodermasuspectum (Eng, J., et al., J. Biol. Chem., 265:20259-62, 1990; Eng.,J., et al., J. Biol. Chem., 267:7402-05, 1992), and agonists, analogs,derivative, variants of either of them as well as biologically activefragments thereof. Exendin-4, as it occurs in the salivary secretions ofthe Gila monster, is an amidated peptide. However, it should beunderstood that the term “exendin,” “exendin-3,” and “exendin-4” referto both the amidated form of the peptide and the acid form of thepeptide. Likewise, reference to GLP-1 generally refers to the amidated7-36 molecule, but it is also intended to include non-amidatedmolecules.

“Exendin agonist” refers to compounds that mimic any effect of anexendin by binding to the receptor or receptors where a naturallyoccurring exendin exerts an effect. Exendin “agonist activity” in thiscontext means having a biological activity of an exendin, such as thosedescribed herein; but it is understood that the activity of the agonistcan be either less potent or more potent than the native exendin.

Exendin-4 is a 39-amino acid polypeptide. Certain sequences are comparedin Table 1.

TABLE 1 a. H A E G T F T S D V S S Y L E G Q A A K E FI A W L V K G R (NH₂) b. H S D G T F T S D L S K Q M E E E A V R L FI E W L K N G G P S S G A P P P S (NH₂)c. D L S K Q M E E E A V R L F I E W L K N G G P S S G A P P P S (NH₂)d. H G E G T F T S D L S K Q M E E E A V R L FI E W L K N G G P S S G A P P P S (NH₂)e. H S D A T F T A E Y S K L L A K L A L Q K YL E S I L G S S T S P R P P S Sf. H S D A T F T A E Y S K L L A K L A L Q K YL E S I L G S S T S P R P P Sg. H S D A I F T E E Y S K L L A K L A L Q K YL A S I L G S R T S P P P (NH₂)h. H S D A I F T Q Q Y S K L L A K L A L Q K YL A S I L G S R T S P P P (NH₂) a = GLP-1(7-36) (NH₂) [SEQ ID NO: 1]. b= exendin 3 (NH₂) [SEQ ID NO: 12]. c = exendin 4 (9-39)(NH₂) [SEQ ID NO:13]. d = exendin 4 (NH₂) [SEQ ID NO: 14]. e = helospectin I [SEQ ID NO:15]. f = helospectin II [SEQ ID NO: 16]. g = helodermin (NH₂) [SEQ IDNO: 17]. h = Q⁸, Q⁹ helodermin (NH₂) [SEQ ID NO: 18].

Various experiments have compared the biologic actions of exendin-4 andGLP-1 and demonstrated a more favorable spectrum of properties forexendin-4. A single subcutaneous dose of exendin-4 lowered plasmaglucose in db/db (diabetic) and ob/ob (diabetic obese) mice by up to40%. In Diabetic Fatty Zucker (ZDF) rats, 5 weeks of treatment withexendin-4 lowered HbA_(1c) (a measure of glycosylated hemoglobin used toevaluate plasma glucose levels) by up to 41%. Insulin sensitivity wasalso improved by 76% following 5 weeks of treatment in obese ZDF rats.In glucose intolerant primates, dose-dependent decreases in plasmaglucose were also observed.

An insulinotropic action of exendin-4 has also been observed in rodents,improving insulin response to glucose by over 100% in non-fasted HarlanSprague Dawley (HSD) rats, and by up to ˜10-fold in non-fasted db/dbmice. Higher pretreatment plasma glucose concentrations were associatedwith greater glucose-lowering effects. Thus the observed glucoselowering effect of exendin-4 appears to be glucose-dependent, andminimal if animals are already euglycemic. Degradation studies withexendin-4 compared to GLP-1 indicate that exendin-4 is relativelyresistant to degradation.

As used in this specification, the term “exendin agonist” includes anymolecules, whether they be peptides, peptide mimetics, or other chemicalcompounds, that bind to or activate a receptor or receptors at whichexendin exerts an effect, as described above. Moreover, exendin agonistsmay include molecules having insulinotropic activity and that may bind aGLP-1 receptor molecule in in vitro assays and induce second messengeractivity on, inter alia, insulin producing β-cells.

The structure activity relationship (SAR) of exendin was investigatedfor structures that may relate to the activity of exendin, for itsstability to metabolism, and for improvement of its physicalcharacteristics, especially as it pertains to peptide stability and toamenability to alternative delivery systems, and various exendin agonistpeptide compounds have been invented. Exendin agonists include exendinanalogs with agonist activity in which one or more naturally ornon-naturally occurring amino acids are added, inserted, eliminated orreplaced with another amino acid(s). Preferred exendin analogs arepeptide analogs of exendin-4.

Exendin analogs include peptides that are encoded by polynucleotidesthat express biologically active exendin analogs with agonist activity,as defined herein. For instance, exendin analogs may be peptidescontaining one or more amino acid substitutions, extensions, additionsor deletions, compared with exendin-4 or exendin-3. In one embodiment,the number of substitutions, extension, deletions, or additions is 30amino acids or less, 25 amino acids or less, 20 amino acids or less, 15amino acids or less, 10 amino acids or less, 5 amino acids or less orany integer in between these amounts. In one aspect of the invention,the substitutions include one or more conservative substitutions.Exendin analogs, which include chemically derivatized or alteredcompounds and peptides having a preferred amino acid homology to SEQ IDNOs:12 and 14 have been previously described and are contemplated to bewithin the scope of the claimed invention.

Novel exendin analogs with agonist activity are described in PCTApplication Serial No. PCT/US98/16387 filed Aug. 6, 1998, entitled“Novel Exendin Agonist Compounds,” which claims the benefit of U.S.Patent Application Ser. No. 60/055,404, filed Aug. 8, 1997, both ofwhich are herein incorporated by reference.

Other novel exendin analogs with agonist activity are described in PCTApplication Serial No. PCT/US98/24210, filed Nov. 13, 1998, entitled“Novel Exendin Agonist Compounds,” which claims the benefit of U.S.Provisional Application No. 60/065,442 filed Nov. 14, 1997, both ofwhich are herein incorporated by reference.

Still other novel exendin analogs with agonist activity are described inPCT Application Serial No. PCT/US98/24273, filed Nov. 13, 1998, entitled“Novel Exendin Agonist Compounds,” which claims the benefit of U.S.Provisional Application No. 60/066,029 filed Nov. 14, 1997, both ofwhich are herein incorporated by reference.

Still other exendin analogs with agonist activity are described in PCTApplication Serial No. PCT/US97/14199, filed Aug. 8, 1997, entitled“Methods for Regulating Gastrointestinal Activity,” which is acontinuation-in-part of U.S. patent application Ser. No. 08/694,954filed Aug. 8, 1996, both of which are hereby incorporated by reference.

Still other exendin analogs with agonist activity are described in PCTApplication Serial No. PCT/US98/00449, filed Jan. 7, 1998, entitled “Useof Exendins and Agonists Thereof for the Reduction of Food Intake,”which claims priority to U.S. Provisional Application No. 60/034,905filed Jan. 7, 1997, both of which are hereby incorporated by reference.

Activity as exendin agonists and exendin analogs with agonist activitycan be indicated, for example, by activity in the assays incorporated byreference in the referenced applications. Effects of exendins or exendinagonists can be identified, evaluated, or screened for, using themethods described herein, or other art-known or equivalent methods fordetermining the effects of exendin. Screening assays for potentialexendin agonist compounds or candidate exendin agonist compounds, mayinclude an in vitro GLP-1 receptor assay/screen described above, anamylin receptor assay/screen using an amylin receptor preparation asdescribed in U.S. Pat. No. 5,264,372, issued Nov. 23, 1993, the contentsof which are incorporated herein by reference, one or more calcitoninreceptor assays/screens using, for example, T47D and MCF7 breastcarcinoma cells, which contain calcium receptors coupled to thestimulation of adenyl cyclase activity, and/or a CGRP receptorassay/screen using, for example, SK-N-MC cells.

Certain preferred exendin analogs with agonist activity include:

exendin-4 (1-30) [SEQ ID NO: 19: His Gly Glu Gly Thr Phe Thr SerAsp Leu Ser Lys Gln Met Glu Glu Glu Ala Val ArgLeu Phe Ile Glu Trp Leu Lys Asn Gly Gly]; exendin-4 (1-30) amide[SEQ ID NO: 20: His Gly Glu Gly Thr Phe Thr SerAsp Leu Ser Lys Gln Met Glu Glu Glu Ala Val ArgLeu Phe Ile Glu Trp Leu Lys Asn Gly Gly-NH₂]; exendin-4 (1-28) amide[SEQ ID NO: 21: His Gly Glu Gly Thr Phe Thr SerAsp Leu Ser Lys Gln Met Glu Glu Glu Ala Val ArgLeu Phe Ile Glu Trp Leu Lys Asn-NH₂]; ¹⁴Leu, ²⁵Phe exendin-4 amide[SEQ ID NO: 22: His Gly Glu Gly Thr Phe Thr SerAsp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val ArgLeu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro SerSer Gly Ala Pro Pro Pro Ser-NH₂]; ¹⁴Leu, ²⁵Phe exendin-4 (1-28) amide[SEQ ID NO: 23: His Gly Glu Gly Thr Phe Thr SerAsp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val ArgLeu Phe Ile Glu Phe Leu Lys Asn-NH₂]; and¹⁴Leu, ²²A1a, ²⁵Phe exendin-4 (1-28) amide[SEQ ID NO: 24: His Gly Glu Gly Thr Phe Thr SerAsp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val ArgLeu Ala Ile Glu Phe Leu Lys Asn-NH₂].

Also included within the scope of the present invention arepharmaceutically acceptable salts of the compounds of formula (III-X)and pharmaceutical compositions including said compounds and saltsthereof.

Formula III

Exendin analogs with agonist activity also include those described inU.S. Provisional Application No. 60/065,442, including compounds of theformula (III) [SEQ ID NO:25]:

Xaa₁ Xaa₂ Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;wherein

Xaa₁ is His, Arg or Tyr;

Xaa₂ is Ser, Gly, Ala or Thr;

Xaa₃ is Ala Asp or Glu;

Xaa₅ is Ala or Thr;

Xaa₆ is Ala, Phe, Tyr or naphthylalanine;

Xaa₇ is Thr or Ser;

Xaa₈ is Ala, Ser or Thr;

Xaa₉ is Asp or Glu;

Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

Xaa₁₁ is Ala or Ser;

Xaa₁₂ is Ala or Lys;

Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

Xaa₁₅ is Ala or Glu;

Xaa₁₆ is Ala or Glu;

Xaa₁₇ is Ala or Glu;

Xaa₁₉ is Ala or Val;

Xaa₂₀ is Ala or Arg;

Xaa₂₁ is Ala or Leu;

Xaa₂₂ is Ala, Phe, Tyr or naphthylalanine;

Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

Xaa₂₆ is Ala or Leu;

Xaa₂₇ is Ala or Lys;

Xaa₂₈ is Ala or Asn;

Z₁ is —OH,

-NH₂ Gly-Z₂, Gly Gly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁ Ser-Z₂,(SEQ ID NO: 35) Gly Gly Xaa₃₁ Ser Ser-Z₂, (SEQ ID NO: 36)Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, (SEQ ID NO 37)Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂, (SEQ ID NO: 38)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, (SEQ ID NO: 39)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ or (SEQ ID NO 40)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;

-   -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro,        homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,        N-alkylpentylglycine or N-alkylalanine; and    -   Z₂ is —OH or —NH₂;

provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala.

Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycine andN-alkylalanine include lower alkyl groups preferably of 1 to about 6carbon atoms, more preferably of 1 to 4 carbon atoms.

Preferred exendin analogs include those wherein Xaa₁ is His or Tyr. Morepreferably Xaa₁ is His.

Preferred are those compounds wherein Xaa₂ is Gly.

Preferred are those compounds wherein Xaa₁₄ is Leu, pentylglycine orMet.

Preferred compounds are those wherein Xaa₂₅ is Trp or Phe.

Preferred compounds are those where Xaa₆ is Phe or naphthylalanine;Xaa₂₂ is Phe or naphthylalanine and

Xaa₂₃ is Ile or Val.

Preferred are compounds wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ areindependently selected from Pro, homoproline, thioproline andN-alkylalanine.

Preferably Z₁ is —NH₂.

Preferably Z₂ is —NH₂.

According to one aspect, preferred are compounds of formula (III)wherein Xaa₁ is His or Tyr, more preferably His; Xaa₂ is Gly; Xaa₆ isPhe or naphthylalanine; Xaa₁₄ is Leu, pentylglycine or Met; Xaa₂₂ is Pheor naphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈are independently selected from Pro, homoproline, thioproline orN-alkylalanine. More preferably Z₁ is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (III) wherein: Xaa₁ is His or Arg;Xaa₂ is Gly or Ala; Xaa₃ is Asp or Glu; Xaa₅ is Ala or Thr; Xaa₆ is Ala,Phe or nephthylalaine; Xaa₇ is Thr or Ser; Xaa₈ is Ala, Ser or Thr; Xaa₉is Asp or Glu; Xaa₁₀ is Ala, Leu or pentylglycine; Xaa₁₁ is Ala or Ser;Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala or Gln; Xaa₁₄ is Ala, Leu orpentylglycine; Xaa₁₅ is Ala or Glu; Xaa₁₆ is Ala or Glu; Xaa₁₇ is Ala orGlu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala or Arg; Xaa₂₁ is Ala or Leu;Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile, Val or tert-butylglycine;Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp or Phe; Xaa₂₆ is Ala or Leu;Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁ is —OH, —NH₂, Gly-Z₂, GlyGly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁ Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser-Z₂(SEQ ID NO:42), Gly Gly Xaa₃₁ Ser Ser Gly-Z₂ (SEQ ID NO:43), Gly GlyXaa₃₁ Ser Ser Gly Ala-Z₂ (SEQ ID NO:44), Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆-Z₂ (SEQ ID NO:45), Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂(SEQ ID NO:46), Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ (SEQID NO:47); Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ being independently Pro,homoproline, thioproline or N-methylalanine; and Z₂ being —OH or —NH₂;provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala. Especially preferredcompounds include those set forth in PCT application Serial No.PCT/US98/24210, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds” identified therein as compounds 2-23.

According to an especially preferred aspect, provided are compoundswhere Xaa₁₄ is Leu, Ile, Val or pentylglycine, more preferably Leu orpentylglycine, and Xaa₂₅ is Phe, Tyr or naphthylalanine, more preferablyPhe or naphthylalanine. These compounds will be less susceptive tooxidative degration, both in vitro and in vivo, as well as duringsynthesis of the compound.

Formula IV

Exendin analogs with agonist activity also include those described inU.S. Provisional Application No. 60/066,029, including compounds of theformula (IV)[SEQ ID NO:26]:

Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;wherein:

Xaa₁ is His, Arg, Tyr, Ala, Norval, Val or Norleu;

Xaa₂ is Ser, Gly, Ala or Thr;

Xaa₃ is Ala, Asp or Glu;

Xaa₄ is Ala, Norval, Val, Norleu or Gly;

Xaa₅ is Ala or Thr;

Xaa₆ is Phe, Tyr or naphthylalanine;

Xaa₇ is Thr or Ser;

Xaa₈ is Ala, Ser or Thr;

Xaa₉ is Ala, Norval, Val, Norleu, Asp or Glu;

Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

Xaa₁₁ is Ala or Ser;

Xaa₁₂ is Ala or Lys;

Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

Xaa₁₅ is Ala or Glu;

Xaa₁₆ is Ala or Glu;

Xaa₁₇ is Ala or Glu;

Xaa₁₉ is Ala or Val;

Xaa₂₀ is Ala or Arg;

Xaa₂₁ is Ala or Leu;

Xaa₂₂ is Phe, Tyr or naphthylalanine;

Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

Xaa₂₆ is Ala or Leu;

Xaa₂₇ is Ala or Lys;

Xaa₂₈ is Ala or Asn;

Z₁ is —OH,

-NH₂, Gly-Z₂, Gly Gly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁ Ser-Z₂,(SEQ ID NO: 35) Gly Gly Xaa₃₁ Ser Ser-Z₂, (SEQ ID NO: 36)Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, (SEQ ID NO: 37)Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂, (SEQ ID NO: 38)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, (SEQ ID NO: 39)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂, (SEQ ID NO: 40)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or (SEQ ID NO: 41)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z₂;

-   -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro,        homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,        N-alkylpentylglycine or N-alkylalanine; and    -   Z₂ is —OH or —NH₂;

provided that no more than three of Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₈, Xaa₉,Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀,Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and provided alsothat, if Xaa₁ is His, Arg or Tyr, then at least one of Xaa₃, Xaa₄ andXaa₉ is Ala.

Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycine andN-alkylalanine include lower alkyl groups preferably of 1 to about 6carbon atoms, more preferably of 1 to 4 carbon atoms. Suitable compoundsof formula (II) include those described in application Serial No.PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds”, identified therein in Examples 1-89 (“Compounds 1-89,”respectively), as well as those corresponding compounds identifiedtherein in Examples 104 and 105.

Preferred such exendin analogs include those wherein Xaa₁ is His, Ala orNorval. More preferably Xaa₁ is His or Ala. Most preferably Xaa₁ is His.

Preferred are those compounds of formula (IV) wherein Xaa₂ is Gly.

Preferred are those compounds of formula (IV) wherein Xaa₃ is Ala.

Preferred are those compounds of formula (IV) wherein Xaa₄ is Ala.

Preferred are those compounds of formula (IV) wherein Xaa₉ is Ala.

Preferred are those compounds of formula (IV) wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (IV) are those wherein Xaa₂₅ is Trp orPhe.

Preferred compounds of formula (IV) are those where Xaa₆ is Ala, Phe ornaphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ is Ile orVal.

Preferred are compounds of formula (IV) wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ andXaa₃₈ are independently selected from Pro, homoproline, thioproline andN-alkylalanine.

Preferably Z₁ is —NH₂.

Preferably Z₂ is —NH₂.

According to one aspect, preferred are compounds of formula (IV) whereinXaa₁ is Ala, His or Tyr, more preferably Ala or His; Xaa₂ is Ala or Gly;Xaa₆ is Phe or naphthylalanine; Xaa₁₄ is Ala, Leu, pentylglycine or Met;Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ are independently selected from Pro, homoproline,thioproline or N-alkylalanine; and Xaa₃₉ is Ser or Tyr, more preferablySer. More preferably Z₁ is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (IV) wherein: Xaa₁ is His or Ala;Xaa₂ is Gly or Ala; Xaa₃ is Ala, Asp or Glu; Xaa₄ is Ala or Gly; Xaa₅ isAla or Thr; Xaa₆ is Phe or naphthylalanine; Xaa₇ is Thr or Ser; Xaa₈ isAla, Ser or Thr; Xaa₉ is Ala, Asp or Glu; Xaa₁₀ is Ala, Leu orpentylglycine; Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ Ala orGln; Xaa₁₄ is Ala, Leu, Met or pentylglycine; Xaa₁₅ is Ala or Glu; Xaa₁₆is Ala or Glu; Xaa₁₇ is Ala or Glu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala orArg; Xaa₂₁ is Ala or Leu; Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile,Val or tert-butylglycine; Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp orPhe; Xaa₂₆ is Ala or Leu; Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁is —OH, —NH₂, Gly-Z₂, Gly Gly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser-Z₂ (SEQ ID NO:42), Gly Gly Xaa₃₁ Ser SerGly-Z₂ (SEQ ID NO:43), Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂ (SEQ ID NO:44),Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂ (SEQ ID NO:45), Gly Gly Xaa₃₁ SerSet Gly Ala Xaa₃₆ Xaa₃₇-Z₂ (SEQ ID NO:46), Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ (SEQ ID NO:47) or Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z₂ (SEQ ID NO:49); Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈being independently Pro, homoproline, thioproline or N-methylalanine;and Z₂ being —OH or —NH₂; provided that no more than three of Xaa₃,Xaa₅, Xaa₆, Xaa₈, Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆,Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ areAla; and provided also that, if Xaa₁ is His, Arg or Tyr, then at leastone of Xaa₃, Xaa₄ and Xaa₉ is Ala. Especially preferred compounds offormula (IV) include those described in application Serial No.PC/7US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds” as having the amino acid sequence of SEQ. ID. NOS. 5-93therein.

According to an especially preferred aspect, provided are compounds offormula (IV) where Xaa₁₄ is Ala, Leu, Ile, Val or pentylglycine, morepreferably Leu or pentylglycine, and Xaa₂₅ is Ala, Phe, Tyr ornaphthylalanine, more preferably Phe or naphthylalanine. These compoundswill be less susceptible to oxidative degration, both in vitro and invivo, as well as during synthesis of the compound.

Formula V

Also within the scope of the present invention are narrower genera ofcompounds having peptides of various lengths, for example genera ofcompounds which do not include peptides having a length of 28, 29 or 30amino acid residues, respectively. Additionally, the present inventionincludes narrower genera of compounds described in PCT applicationSerial No. PCT/US98/24210, filed Nov. 13, 1998, entitled “Novel ExendinAgonist Compounds” and having particular amino acid sequences, forexample, compounds of the formula (V) [SEQ. ID. NO:27]:

Xaa₁ Xaa₂ Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇Ala Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;wherein:

Xaa₁ is His or Arg;

Xaa₂ is Gly or Ala;

Xaa₃ is Ala, Asp or Glu;

Xaa₅ is Ala or Thr;

Xaa₆ is Ala, Phe or naphthylalanine;

Xaa₇ is Thr or Ser;

Xaa₈ is Ala, Ser or Thr;

Xaa₉ is Asp or Glu;

Xaa₁₀ is Ala, Leu or pentylglycine;

Xaa₁₁ is Ala or Ser;

Xaa₁₂ is Ala or Lys;

Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu or pentylglycine;

Xaa₁₅ is Ala or Glu;

Xaa₁₆ is Ala or Glu;

Xaa₁₇ is Ala or Glu;

Xaa₁₉ is Ala or Val;

Xaa₂₀ is Ala or Arg;

Xaa₂₁ is Ala or Leu;

Xaa₂₂ is Phe or naphthylalanine;

Xaa₂₃ is Ile, Val or tert-butylglycine;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp, or Phe;

Xaa₂₆ is Ala or Leu;

Xaa₂₇ is Ala or Lys;

Xaa₂₈ is Ala or Asn;

Z₁ is —OH,

-NH₂, Gly-Z₂, Gly Gly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁ Ser-Z₂,(SEQ ID NO: 42) Gly Gly Xaa₃₁ Ser Ser-Z₂, (SEQ ID NO: 43)Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, (SEQ ID NO: 44)Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂, (SEQ ID NO: 45)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, (SEQ ID NO: 46)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂; or (SEQ ID NO: 47)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;

-   -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from        the group consisting of Pro, homoproline, thioproline and        N-methylylalanine; and    -   Z₂ is —OH or —NH₂;

provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and pharmaceuticallyacceptable salts thereof.

Formula VI

Additionally, the present invention includes narrower genera of peptidecompounds described in PCT Application Serial No. PCT/US98/24273, filedNov. 13, 1998, entitled “Novel Exendin Agonist Compounds” as havingparticular amino acid sequences, for example, compounds of the formula[VI] [SEQ. ID. NO:28]:

Xaa₁ Xaa₂ Xaa₃ Xaa₅ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇Ala Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;wherein:

Xaa₁ is His or Ala;

Xaa₂ is Gly or Ala;

Xaa₃ is Ala, Asp or Glu;

Xaa₄ is Ala or Gly;

Xaa₅ is Ala or Thr;

Xaa₆ is Phe or naphthylalanine;

Xaa₇ is Thr or Ser;

Xaa₈ is Ala, Ser or Thr;

Xaa₉ is Ala, Asp or Glu;

Xaa₁₀ is Ala, Leu or pentylglycine;

Xaa₁₁ is Ala or Ser;

Xaa₁₂ is Ala or Lys;

Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Met or pentylglycine;

Xaa₁₅ is Ala or Glu;

Xaa₁₆ is Ala or Glu;

Xaa₁₇ is Ala or Glu;

Xaa₁₉ is Ala or Val;

Xaa₂₀ is Ala or Arg;

Xaa₂₁ is Ala or Leu;

Xaa₂₂ is Phe or naphthylalanine;

Xaa₂₃ is Ile, Val or tert-butylglycine;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp or Phe;

Xaa₂₆ is Ala or Leu;

Xaa₂₇ is Ala or Lys;

Xaa₂₈ is Ala or Asn;

Z₁ is —OH,

-NH₂, Gly-Z₂, Gly Gly-Z₂ Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁ Ser-Z₂,(SEQ ID NO: 42) Gly Gly Xaa₃₁ Ser Ser-Z₂ (SEQ ID NO: 43)Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, (SEQ ID NO: 44)Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂, (SEQ ID NO: 45)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, (SEQ ID NO: 46)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ (SEQ ID NO: 47)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ (SEQ ID NO: 48)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Ser-Z₂;

-   -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro,        homoproline, thioproline, or N-methylylalanine; and    -   Z₂ is —OH or —NH₂;

provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇, and Xaa₂₈ are Ala; and provided that, ifXaa₁ is His, Arg or Tyr, then at least one of Xaa₃, Xaa₄ and Xaa₉ isAla; and pharmaceutically acceptable salts thereof.

Preferred compounds of formula (VI) include those wherein Xaa₁ is His,Ala, Norval or 4-imidazopropionyl. Preferably, Xaa₁ is His, or4-imidazopropionyl or Ala, more preferably His or 4-imidazopropionyl.

Preferred compounds of formula (VI) include those wherein Xaa2 is Gly.

Preferred compounds of formula (VI) include those wherein Xaa4 is Ala.

Preferred compounds of formula (VI) include those wherein Xaa9 is Ala.

Preferred compounds of formula (VI) include those wherein Xaa14 is Leu,pentylglycine or Met.

Preferred compounds of formula (VI) include those wherein Xaa25 is Trpor Phe.

Preferred compounds of formula (VI) include those wherein Xaa6 is Ala,Phe or naphthylalanine; Xaa22 is Phe or naphthylalanine; and Xaa23 isIle or Val.

Preferred compounds of formula (VI) include those wherein Z1 is —NH2.

Preferred compounds of formula (VI) include those wherein Xaa31, Xaa36,Xaa37 and Xaa38 are independently selected from the group consisting ofPro, homoproline, thioproline and N-alkylalanine.

Preferred compounds of formula (VI) include those wherein Xaa39 is Seror Tyr, preferably Ser.

Preferred compounds of formula (VI) include those wherein Z2 is —NH2.

Preferred compounds of formula (VI) include those 42 wherein Z1 is —NH2.

Preferred compounds of formula (VI) include those wherein Xaa21 isLys-NH2-R where R is Lys, Arg, C1-C10 straight chain or branchedalkanoyl.

Preferred compounds of formula (VI) include those wherein X1 is Lys Asn,Lys-NHε-R Asn, or Lys-NHε-R Ala where R is Lys, Arg, C1-C10 straightchain or branched alkanoyl. Preferred compounds of formula (VI) includethose having an amino acid sequence described in PCT application SerialNo. PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds” as being selected from SEQ. ID. NOS. 95-110 therein.

Formula VII

Also provided are compounds described in PCT application PCT/US98/24210,filed Nov. 13, 1998, entitled “Novel Exendin Agonist Compounds”,including compounds of the formula (VII) [SEQ. ID. NO. 29]:

Xaa₁ Xaa₂ Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ X₁-Z₁;wherein

Xaa₁ is His, Arg or Tyr or 4-imidazopropionyl;

Xaa₂ is Ser, Gly, Ala or Thr;

Xaa₃ is Ala, Asp or Glu;

Xaa₅ is Ala or Thr;

Xaa₆ is Ala, Phe, Tyr or naphthylalanine;

Xaa₇ is Thr or Ser;

Xaa₈ is Ala, Ser or Thr;

Xaa₉ is Asp or Glu;

Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

Xaa₁₁ is Ala or Ser;

Xaa₁₂ is Ala or Lys;

Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

Xaa₁₅ is Ala or Glu;

Xaa₁₆ is Ala or Glu;

Xaa₁₇ is Ala or Glu;

Xaa₁₉ is Ala or Val;

Xaa₂₀ is Ala or Arg;

Xaa₂₁ is Ala, Leu or Lys-NH^(ε)-R where R is Lys, Arg, C₁-C₁₀ straightchain or branched alkanoyl or cycloalkylalkanoyl;

Xaa₂₂ is Phe, Tyr or naphthylalanine;

Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

Xaa₂₆ is Ala or Leu;

X₁ is Lys Asn, Asn Lys, Lys-NH^(ε)-R Asn, Asn Lys-NH^(ε)-R, Lys-NH^(ε)-RAla, Ala Lys-NH^(ε)-R where R is Lys, Arg, C₁-C₁₀ straight chain orbranched alkanoyl or cycloalkylalkanoyl

Z₁ is —OH,

-NH₂, Gly-Z₂, Gly Gly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁ Ser-Z₂,(SEQ ID NO: 35) Gly Gly Xaa₃₁ Ser Ser-Z₂, (SEQ ID NO: 36)Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, (SEQ ID NO: 37)Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂, (SEQ ID NO: 38)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, (SEQ ID NO: 39)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ or (SEQ ID NO: 40)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;

-   -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from        the group consisting of Pro, homoproline, 3Hyp, 4Hyp,        thioproline, N-alkylglycine, N-alkylpentylglycine and        N-alkylalanine; and    -   Z₂ is —OH or —NH₂;

provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, and Xaa₂₆ are Ala. Also within the scope of the presentinvention are pharmaceutically acceptable salts of the compound offormula (VII) and pharmaceutical compositions including said compoundsand salts thereof.

Preferred exendin analogs of formula (VII) include those wherein Xaa₁ isHis, Tyr or 4-imidazopropionyl. More preferably Xaa₁ is His.

Preferred are those compounds of formula (VII) wherein Xaa₁ is4-imidazopropionyl.

Preferred are those compounds of formula (VII) wherein Xaa₂ is Gly.

Preferred compounds of formula (VII) are those wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (VII) are those wherein Xaa₂₅ is Trp orPhe.

According to one aspect, preferred are compounds of formula (VII)wherein Xaa₆ is Phe or naphthylalanine; and Xaa₂₂ is Phe ornaphthylalanine; and Xaa₂₃ is Ile or Val. More preferably, Z₁ is —NH₂.According to one aspect, especially preferred are such compounds offormula (VII) wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independentlyselected from the group consisting of Pro, homoproline, thioproline andN-alkylalanine. More preferds, Z₂ is —NH₂.

Preferred compounds of formula (VII) include those wherein X₁ is LysAsn, Lys-NH^(ε)-R Asn, or Lys-NH^(ε)-R Ala where R is Lys, Arg, C₁-C₁₀straight chain or branched alkanoyl. Preferred compounds of formula(VII) include compounds described in PCT application Serial No.PCT/US98/24210, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds” and identified therein as Compound Nos. 62-69.

Preferred such exendin analogs include those wherein Xaa₁ is His, Ala orNorval. More preferably Xaa₁ is His or Ala. Most preferably Xaa₁ is His.

Preferred are those compounds of formula (VII) wherein Xaa₂ is Gly.

Preferred are those compounds of formula (VII) wherein Xaa₃ is Ala.

Preferred are those compounds of formula (VII) wherein Xaa₄ is Ala.

Preferred are those compounds of formula (VII) wherein Xaa₉ is Ala.

Preferred are those compounds of formula (VII) wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (VII) are those wherein Xaa₂₅ is Trp orPhe.

Preferred compounds of formula (VII) are those where Xaa₆ is Ala, Phe ornaphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ is Ile orVal.

Preferred are compounds of formula (VII) wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ andXaa₃₈ are independently selected from Pro, homoproline, thioproline andN-alkylalanine.

Preferably Z₁ is —NH₂.

Preferably Z₂ is —NH₂.

According to one aspect, preferred are compounds of formula (VII)wherein Xaa₁ is Ala, His or Tyr, more preferably Ala or His; Xaa₂ is Alaor Gly; Xaa₆ is Phe or naphthylalanine; Xaa₁₄ is Ala, Leu, pentylglycineor Met; Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁,Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from Pro, homoproline,thioproline or N-alkylalanine; and Xaa₃₉ is Ser or Tyr, more preferablySer. More preferably Z₁ is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (VII) wherein: Xaa₁ is His or Ala;Xaa₂ is Gly or Ala; Xaa₃ is Ala, Asp or Glu; Xaa₄ is Ala or Gly; Xaa₅ isAla or Thr; Xaa₆ is Phe or naphthylalanine; Xaa₇ is Thr or Ser; Xaa₈ isAla, Ser or Thr; Xaa₉ is Ala, Asp or Glu; Xaa₁₀ is Ala, Leu orpentylglycine; Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala orGln; Xaa₁₄ is Ala, Leu, Met or pentylglycine; Xaa₁₅ is Ala or Glu; Xaa₁₆is Ala or Glu; Xaa₁₇ is Ala or Glu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala orArg; Xaa₂₁ is Ala or Leu; Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile,Val or tert-butylglycine; Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp orPhe; Xaa₂₆ is Ala or Leu; Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁is —OH, —NH₂, Gly-Z₂, Gly Gly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser-Z₂ (SEQ ID NO:42), Gly Gly Xaa₃, Ser SerGly-Z₂ (SEQ ID NO:43), Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂ (SEQ ID NO:44),Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂ (SEQ ID NO:45), Gly Gly Xaa₃₁ SerSer Gly Ala Xaa₃₆ Xaa₃₇-Z₂ (SEQ ID NO:46), Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ (SEQ ID NO:47) or Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z₂ (SEQ ID NO:49); Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈being independently Pro, homoproline, thioproline or N-methylalanine;and Z₂ being —OH or —NH₂; provided that no more than three of Xaa₃,Xaa₅, Xaa₆, Xaa₈, Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆,Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ areAla; and provided also that, if Xaa₁ is His, Arg or Tyr, then at leastone of Xaa₃, Xaa₄ and Xaa₉ is Ala. Especially preferred compounds offormula (VII) include those described in POT application Serial No.PCT/US98/24210, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds” and having the amino acid sequences identified therein asSEQ. ID. NOS. 5-93.

According to an especially preferred aspect, provided are compounds offormula (VII) where Xaa14 is Ala, Leu, Ile, Val or pentylglycine, morepreferably Leu or pentylglycine, and Xaa25 is Ala, Phe, Tyr ornaphthylalanine, more preferably Phe or naphthylalanine. These compoundswill be less susceptible to oxidative degration, both in vitro and invivo, as well as during synthesis of the compound.

Formula VIII

Also provided are peptide compounds described in PCT Application SerialNo. PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds”, including compounds of the formula (VIII) [SEQ. ID. NO:30]:

Xaa₁ Xaa₂ Xaa₃ Xaa4 Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ X₁-Z₁;wherein

Xaa₁ is His, Arg, Tyr, Ala, Norval, Val, Norleu or 4-imidazopropionyl;

Xaa₂ is Ser, Gly, Ala or Thr;

Xaa₃ is Ala, Asp or Glu;

Xaa₄ is Ala, Norval, Val, Norleu or Gly;

Xaa₅ is Ala or Thr;

Xaa₆ is Phe, Tyr or naphthylalanine;

Xaa₇ is Thr or Ser;

Xaa₈ is Ala, Ser or Thr;

Xaa₉ is Ala, Norval, Val, Norleu, Asp or Glu;

Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

Xaa₁₁ is Ala or Ser;

Xaa₁₂ is Ala or Lys;

Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

Xaa₁₅ is Ala or Glu;

Xaa₁₆ is Ala or Glu;

Xaa₁₇ is Ala or Glu;

Xaa₁₉ is Ala or Val;

Xaa₂₀ is Ala or Arg;

Xaa₂₁ is Ala, Leu or Lys-NH^(ε)-R where R is Lys, Arg, C¹⁻¹⁰ straightchain or branched alkanoyl or cycloalleyl-alkanoyl;

Xaa₂₂ is Phe, Tyr or naphthylalanine;

Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

Xaa₂₆ is Ala or Leu;

X₁ is Lys Asn, Asn Lys, Lys-NH^(ε)-R Asn, Asn Lys-NH^(ε)-R, Lys-NH^(ε)-RAla, Ala Lys-NH^(ε)-R where R is Lys, Arg, C₁-C₁₀ straight chain orbranched alkanoyl or cycloalkylalkanoyl

Z₁ is —OH,

-NH₂, Gly-Z₂, Gly Gly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁ Ser-Z₂,(SEQ ID NO: 35) Gly Gly Xaa₃₁ Ser Ser-Z₂, (SEQ ID NO: 36)Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, (SEQ ID NO: 37)Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂, (SEQ ID NO: 38)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, (SEQ ID NO: 39)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂, (SEQ ID NO: 40)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or (SEQ ID NO: 41)Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z₂;;

-   -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from        the group consisting of Pro, homoproline, 3Hyp, 4Hyp,        thioproline, N-alkylglycine, N-alkylpentylglycine and        N-alkylalanine; and    -   Z₂ is —OH or —NH₂;

provided that no more than three of Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₈, Xaa₉,Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀,Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, are Ala; and provided also that, if Xaa₁ isHis, Arg, Tyr, or 4-imidazopropionyl then at least one of Xaa₃, Xaa₄ andXaa₉ is Ala.

Preferred compounds of formula (VIII) include those wherein Xaa₁ is His,Ala, Norval or 4-imidazopropionyl. Preferably, Xaa₁ is His, or4-imidazopropionyl or Ala, more preferably His or 4-imidazopropionyl.

Preferred compounds of formula (VIII) include those wherein Xaa₂ is Gly.

Preferred compounds of formula (VIII) include those wherein Xaa₄ is Ala.

Preferred compounds of formula (VIII) include those wherein Xaa₉ is Ala.

Preferred compounds of formula (VIII) include those wherein Xaa₁₄ isLeu, pentylglycine or Met.

Preferred compounds of formula (VIII) include those wherein Xaa₂₅ is Trpor Phe.

Preferred compounds of formula (VIII) include those wherein Xaa₆ is Ala,Phe or naphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ isIle or Val.

Preferred compounds of formula (VIII) include those wherein Z₁ is —NH₂.

Preferred compounds of formula (VIII) include those wherein Xaa₃₁,Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from the groupconsisting of Pro, homoproline, thioproline and N-alkylalanine.

Preferred compounds of formula (VIII) include those wherein Xaa₃₉ is Seror Tyr, preferably Ser.

Preferred compounds of formula (VIII) include those wherein Z₂ is —NH₂.

Preferred compounds of formula (VIII) include those 42 wherein Z₁ is—NH₂.

Preferred compounds of formula (VIII) include those wherein Xaa₂₁ isLys-NH^(ε)-R where R is Lys, Arg, C₁-C₁₀ straight chain or branchedalkanoyl.

Preferred compounds of formula (VIII) include those wherein X₁ is LysAsn, Lys-NH^(ε)-R Asn, or Lys-NH^(ε)-R Ala where R is Lys, Arg, C₁-C₁₀straight chain or branched alkanoyl.

Preferred compounds of formula (VIII) include those described in PCTApplication Serial No. PCT/US98/24273, filed Nov. 13, 1998, entitled“Novel Exendin Agonist Compounds” as having an amino acid sequenceselected from those identified therein as SEQ. ID. NOS. 95-110.

Formula IX

Compounds particularly useful according to the present invention areexendin analogs with agonist activity described in U.S. patentapplication Ser. No. 09/003,869, filed Jan. 7, 1998, entitled “Use ofExendins And Agonists Thereof For The Reduction of Food Intake”,including compounds of the formula (IX) [SEQ. ID. NO:31]:

Xaa₁ Xaa₂ Xaa₃ Gly Thr Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈Ser Lys Gln Xaa₉ Glu Glu Glu Ala Val Arg LeuXaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Leu Lys Asn Gly Gly Xaa₁₄Ser Ser Gly Ala Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈-Zwherein:

Xaa₁ is His, Arg or Tyr;

Xaa₂ is Ser, Gly, Ala or Thr;

Xaa₃ is Asp or Glu;

Xaa₄ is Phe, Tyr or naphthalanine;

Xaa₅ is Thr or Ser;

Xaa₆ is Ser or Thr;

Xaa₇ is Asp or Glu;

Xaa₈ is Leu, Ile, Val, pentylglycine or Met;

Xaa₉ is Leu, Ile, pentylglycine, Val or Met;

Xaa₁₀ is Phe, Tyr or naphthalanine;

Xaa₁₁ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₁₂ is Glu or Asp; Xaa₁₃ is Trp, Phe, Tyr, or naphthylalanine;

Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently Pro, homoproline, 3Hyp,4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine orN-alkylalanine;

Xaa₁₈ is Ser, Thr or Tyr; and Z is —OH or —NH₂;

with the proviso that the compound does not have the formula of eitherSEQ. ID. NOS:12 or 14. Preferred N-alkyl groups for N-alkylglycine,N-alkylpentylglycine and N-alkylalanine include lower alkyl groupspreferably of 1 to about 6 carbon atoms, more preferably of 1 to 4carbon atoms. Also useful in the present invention are pharmaceuticallyacceptable salts of the compounds of formula (IX).

Preferred exendin analogs include those wherein Xaa₁ is His or Tyr. Morepreferably Xaa₁ is His.

Preferred are those compounds wherein Xaa₂ is Gly.

Preferred are those compounds wherein Xaa₉ is Leu, pentylglycine or Met.

Preferred compounds include those wherein Xaa₁₃ is Trp or Phe.

Also preferred are compounds where Xaa₄ is Phe or naphthalanine; Xaa₁₁is Ile or Val and Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independentlyselected from Pro, homoproline, thioproline or N-alkylalanine.Preferably N-alkylalanine has a N-alkyl group of 1 to about 6 carbonatoms.

According to an especially preferred aspect, Xaa₁₅, Xaa₁₆ and Xaa₁₇ arethe same amino acid reside.

Preferred are compounds wherein Xaa₁₈ is Ser or Tyr, more preferablySer.

Preferably Z is —NH₂.

According to one aspect, preferred are compounds of formula (VII)wherein Xaa₁ is His or Tyr, more preferably His; Xaa₂ is Gly; Xaa₄ isPhe or naphthalanine; Xaa₉ is Leu, pentylglycine or Met; Xaa₁₀ is Phe ornaphthalanine; Xaa₁₁ is Ile or Val; Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ areindependently selected from Pro, homoproline, thioproline orN-alkylalanine; and Xaa₁₈ is Ser or Tyr, more preferably Ser. Morepreferably Z is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (IX) wherein: Xaa₁ is His or Arg;Xaa₂ is Gly; Xaa₃ is Asp or Glu; Xaa₄ is Phe or napthylalanine; Xaa₅ isThr or Ser; Xaa₆ is Ser or Thr; Xaa₇ is Asp or Glu; Xaa₈ is Leu orpentylglycine; Xaa₉ is Leu or pentylglycine; Xaa₁₀ is Phe ornaphthylalanine; Xaa₁₁ is Ile, Val or t-butyltylglycine; Xaa₁₂ is Glu orAsp; Xaa₁₃ is Trp or Phe; Xaa₁₄, Xaa₁₅, Xaa₁₆, and Xaa₁₇ areindependently Pro, homoproline, thioproline, or N-methylalanine; Xaa₁₈is Ser or Tyr: and Z is —OH or —NH₂; with the proviso that the compounddoes not have the formula of either SEQ. ID. NOS. 7 or 9. Morepreferably Z is —NH₂.

According to an especially preferred aspect, provided are compoundswhere Xaa₉ is Leu, Ile, Val or pentylglycine, more preferably Leu orpentylglycine, and Xaa₁₃ is Phe, Tyr or naphthylalanine, more preferablyPhe or naphthylalanine. These compounds are believed to exhibitadvantageous duration of action and to be less subject to oxidativedegration, both in vitro and in vivo, as well as during synthesis of thecompound.

Formula X

Also provided are compounds described in PCT Application Serial No.PCT/US98/16387, filed Aug. 6, 1998, entitled “Novel Exendin AgonistCompounds”, including compounds of the formula (X) [SEQ. ID. NO:32]:

Xaa₁ Xaa₂ Xaa₃ Gly Thr Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈Ser Lys Gln Xaa₉ Glu Glu Glu Ala Val Arg LeuXaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Leu X₁ Gly Gly Xaa₁₄Ser Ser Gly Ala Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈-Zwherein:

Xaa₁ is His, Arg, Tyr or 4-imidazopropionyl;

Xaa₂ is Ser, Gly, Ala or Thr;

Xaa₃ is Asp or Glu;

Xaa₄ is Phe, Tyr or naphthylalanine;

Xaa₅ is Thr or Ser;

Xaa₆ is Ser or Thr;

Xaa₇ is Asp or Glu;

Xaa₈ is Leu, Ile, Val, pentylglycine or Met;

Xaa₉ is Leu, Ile, pentylglycine, Val or Met;

Xaa₁₀ is Phe, Tyr or naphthylalanine;

Xaa₁₁ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₁₂ is Glu or Asp;

Xaa₁₃ is Trp, Phe, Tyr, or naphthylalanine; X₁ is Lys Asn, Asn Lys,Lys-NH^(ε)-R Asn, Asn Lys-NH^(ε)-R where R is Lys, Arg, C₁-C₁₀ straightchain or branched alkanoyl or cycloalkylalkanoyl;

Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently Pro, homoproline, 3Hyp,4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine orN-alkylalanine;

Xaa₁₈ is Ser, Thr or Tyr; and Z is —OH or —NH₂;

with the proviso that the compound does not have the formula of eitherSEQ. ID. NOS. 7 or 9. Suitable compounds of formula (X) includecompounds described in PCT Application Serial No. PCT/US98/16387, filedAug. 6, 1998, entitled “Novel Exendin Agonist Compounds” having theamino acid sequences of SEQ. ID. NOS. 37-40 therein.

Preferred exendin analogs of formula (X) include those wherein Xaa₁ isHis, Tyr or 4-imidazopropionyl. More preferably, Xaa₁ is His or4-imidazopropionyl.

Preferred are those compounds of formula (X) wherein Xaa₂ is Gly.

Preferred are those compounds of formula (X) wherein Xaa₉ is Leu,pentylglycine or Met.

Preferred are those compounds of formula (X) wherein Xaa₁₃ is Trp orPhe.

Preferred are those compounds of formula (X) wherein

X₁ is Lys Asn, or Lys-NH^(ε)-R Asn, where R is Lys, Arg, C₁-C₁₀ straightchain or branched alkanoyl.

Also preferred are compounds of formula (X) wherein Xaa₄ is Phe ornaphthylalanine; Xaa₁₀ is Phe or naphthylalanine; Xaa₁₁ is Ile or Valand Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently selected from Pro,homoproline, thioproline or N-alkylalanine. According to an especiallypreferred aspect, Xaa₁₈ is Ser or Tyr. Preferred are those suchcompounds wherein Xaa₁₈ is Ser. Preferably, Z is —NH₂.

According to one preferred aspect, preferred are compounds of formula(X) wherein Xaa₄ is Phe or naphthylalanine; Xaa₁₀ is Phe ornaphthylalanine; Xaa₁₁ is Ile or Val, X₁ is Lys Asn, or Lys-NH^(ε)-RAsn, where R is Lys, Arg, straight chain or branched alkanoyl and Xaa₁₄,Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently selected from Pro, homoproline,thioproline or N-alkylalanine.

Exendins and exendin agonists that are peptides, such as exendinanalogs, described herein may be prepared through peptide purificationas described in, for example, Eng, et al., J. Biol. Chem. 265:20259-62,1990; and Eng, et al., J. Biol. Chem. 267:7402-05, 1992, herebyincorporated by reference herein. Alternatively, exendins and exendinagonists that are peptides may be prepared by methods known to thoseskilled in the art, for example, as described in Raufman, et al., J.Biol. Chem. 267:21432-37, 1992), hereby incorporated by referenceherein, using standard solid-phase peptide synthesis techniques andpreferably an automated or semiautomated peptide synthesizer aspreviously described and is well known in the art.

Exendins and exendin agonists that are peptides may also be preparedusing recombinant DNA techniques, using methods now known in the art.See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2dEd., Cold Spring Harbor (1989). Alternatively, such compounds may beprepared by homogeneous phase peptide synthesis methods. Non-peptidecompounds useful in the present invention may be prepared by art-knownmethods. For example, phosphate-containing amino acids and peptidescontaining such amino acids, may be prepared using methods known in theart. See, e.g., Bartlett and Landen, Biorg. Chem. 14:356-377 (1986).Methods for making and/or purifying GLP-1 and its agonists, analogs,derivatives, variants, and fragments, as discussed previously, can alsobe utilized to make and/or purify exendins, their agonists, analogs,derivatives, variants, and fragments thereof.

The compositions of the present invention may be used in combinationwith a suitable pharmaceutical carrier. Such compositions comprise atherapeutically effective amount of the polypeptide, and apharmaceutically acceptable carrier or excipient. The compositions ofthis invention can be administered in any effective, pharmaceuticallyacceptable form for warm blooded animals, including human and otheranimal subjects, e.g., in topical, lavage, oral, suppository,parenteral, or infusible dosage forms, as a topical, buccal, sublingual,pulmonary, or nasal spray or in any other manner effective to deliverthe agents. The route of administration will preferably be designed tooptimize delivery and/or localization of the agents.

In addition to the active compositions of the invention, thepharmaceutical composition may contain suitable excipients andauxiliaries that facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Oral dosage formsencompass tablets, capsules, granules, solutions, and suspensions.Preparations that can be administered rectally include suppositories.Other dosage forms include suitable solutions for administrationparenterally or orally, and compositions which can be administeredbuccally or sublingually.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself well known in the art. Forexample the pharmaceutical preparations may be made by means ofconventional mixing, granulating, dissolving, and lyophilizingprocesses. The processes to be used will depend ultimately on thephysical properties of the active ingredient used.

Suitable formulations for parenteral administration include aqueoussolutions of active compounds in water-soluble or water-dispersibleform. In addition, suspensions of the active compounds as appropriateoily injection suspensions may be administered. Suitable lipophilicsolvents or vehicles include fatty oils for example, sesame oil, orsynthetic fatty acid esters, for example, ethyl oleate or triglycerides.Aqueous injection suspensions may contain substances that increase theviscosity of the suspension, including for example, sodium carboxymethylcellulose, sorbitol and/or dextran. Such compositions may also compriseadjuvants such as preserving, wetting, emulsifying, and dispensingagents. They may also be sterilized, for example, by filtration througha bacteria-retaining filter, or by incorporating sterilizing agents intothe compositions. They can also be manufactured in the form of sterilesolid compositions that can be dissolved or suspended in sterile water,saline, or other injectable medium prior to administration.

In addition to administration with conventional carriers, activeingredients may be administered by a variety of specialized deliverydrug techniques that are known to those of skill in the art, such asportable infusion pumps.

Additional formulations for administration may be made in accordancewith methods and amounts known in the art as set forth in Remington'sPharmaceutical Sciences, 18th Ed., Wiley Publishing (1990), thedisclosure of which is herein incorporated by references in itsentirety.

The compositions of the present invention can be administered along witha pharmaceutically acceptable carrier in an amount sufficient to preventarrhythmias and/or treat an active arrhythmia. The compounds of thisinvention have extremely low toxicity and a low degree of side effectseven at high doses. The dosing range of the compounds of this inventionwill vary depending on a number of factors, such as whether it is usedfor prophylaxis or treatment of arrhythmia, route of administration,desired dosing schedule, the physical health of the patient, etc.

Although not limited to the following ranges and provided only as anillustration, exemplary dose ranges for use in the invention can include0.001 pmol/kg to 500 nmol/kg per day depending on the compositionselected. A lower limit of a dosage range can be about 0.001 pmol/kg,0.01 pmol/kg, 0.1 pmol/kg, 1 pmol/kg, 10 pmol/kg, or 100 pmol/kg. Anupper dosage range can be about 10 pmol/kg, 100 pmol/kg, 1 nmol/kg, 10nmol/kg, 100 nmol/kg, 250 nmol/kg or 500 nmol/kg. The desired dose willvary depending on the selected active composition. The desired dose willalso depend upon other factors including the route of administration andthe formulation. For example, continuous infusion as well as bolus dosesand sustained release formulations are contemplated. Routes ofadministration include intramuscular, intravenous, subcutaneous,intradermal, transdermal, intraarticular, intrathecal and the like.Mucosal delivery is also contemplated. These routes include, but are notlimited to, oral, nasal, sublingual, rectal, pulmonary and buccalroutes, which may include administration of the peptide in liquid,semi-solid or solid form.

Exemplary doses for continuous infusion by intravenous (I.V.) can beabout 0.1 pmol/kg/min to 10 pmol/kg/min and by subcutaneous (s.c.) about0.1 pmol/kg/min to 75 pmol/kg/min., and for single injection (bolus) byI.V. about 0.1 nmol/kg to 2.0 nmol/kg and s.c. about 0.1 nmol/kg to 100nmol/kg. The foregoing doses may be administered as a single dose or maybe divided into multiple doses for administration. The peptides of thisinvention may be administered once to several times daily.

While a preferred method of administration of a GLP-1 peptide may bethrough a continuous application, other forms of delivery as describedabove are also contemplated. However, an exemplary dosing rate can bewithin a range of from about 1 to about 10 pmol/kg per minute of GLP-1delivered by sustained release subcutaneous, intramuscular,interperitoneal, injected depot with sustained release, deep lunginsufflation, as well as by intravenous, buccal, patch or othersustained release delivery methods. Degradation-resistant GLP-1 analogs,derivatives or variants, exendins, analogs, derivatives or variants, andother molecules of the invention need not be delivered continuously, butare suitable for bolus or sustained release dosing and may be at dosesmuch lower than those described.

Other drugs besides compositions of the invention which are compatiblewith the carrier ingredients may also be incorporated into thepharmaceutical formulations. Such drugs may be readily ascertained bythose of ordinary skill in the art and may include, for instance,anti-inflammatory agents, diuretics, vasodilators, etc.

It is understood that the present invention contemplates the use of notonly the above-stated active forms of the compositions of the invention,but also includes the prodrugs (proforms) which metabolize to thecompound and biologically active salt forms thereof, as well as opticalisomers which provide the same pharmaceutical results.

The compositions of the invention may also be used in combination withagents known in the art that enhance the half-life in vivo of peptide inorder to enhance or prolong the biological activity of the peptide. Forexample, a molecule or chemical moiety may be covalently linked to thecomposition of the present invention before administration thereof.Alternatively, the enhancing agent may be administered concurrently withthe composition. Still further, the agent may comprise a molecule thatis known to inhibit the enzymatic degradation of the compositions of theinvention that may be administered concurrently with or afteradministration of the composition. Such a molecule may be administered,for example, orally, by injection, or any other means known in the art.

In accordance with this invention, compositions of the invention incombination with a pharmaceutically acceptable carrier are preferablyadministered within the first four hours following an ischemic event inorder to prevent the occurrence of cardiac arrhythmia. Compositions ofthe invention can be co-administered with glucose (5%) if required tomaintain blood glucose levels ≧5 mM (to maintain efficient insulinsecretion). Similarly, co-administration of potassium (K⁺) may beconsidered, depending on the extent to which activation of the membraneNa⁺/K⁺ ATPase leads to a shift of K⁺ into the intracellular space.

With respect to reperfusion, treatment with compositions of theinvention should be commenced concurrently or as soon as possiblefollowing therapies that reestablish flow in an artery that wasobstructed by a blood clot (e.g., thromolytic therapy) or otherobstructive materials, or following an intervention, such asangioplasty, coronary bypass grafting, or placement of an intracoronarystent. Therapy should continue thereafter. In the case of cardiacsurgery, the treatment should preferably commence 12-24 hours prior tosurgery, during surgery from the onset of anesthesia until aorticcrossclamping, and immediately after unclamping for a period of at least72 hours postoperatively. As earlier explained, co-administration of afree radical scavenger or antioxidants will further aid reperfusionrecovery.

We claim:
 1. A method for treating arrhythmias, the method comprising:administering to a subject in need of such treatment an amount of acomposition effective to treat arrhythmias, said composition comprisinga polypeptide of Formula VII (SEQ ID NO:29) or VIII (SEQ ID NO:30) thatbinds to a receptor for GLP-1; and thereby treating said arrhythmias,wherein Xaa₁ is 4-imidazopropionyl; Xaa₂₁ is Lys-NH^(ε)-R where R isLys, Arg, C₁-C₁₀ straight chain or branched chain alkanoyl orcycloalkylalkanoyl; and X₁ is Lys Asn, Asn Lys, Lys-NH^(ε)-R Asn, AsnLys-NH^(ε)-R, Lys-NH^(ε)-R Ala, Ala Lys-NH^(ε)-R where R is Lys, Arg,C₁-C₁₀ straight chain or branched chain alkanoyl or cycloalkylalkanoyl.2. The method of claim 1 wherein the composition is administered in adose of from about 0.1 pmol/kg/min. up to about 10 pmol/kg/min.
 3. Themethod of claim 1 wherein the composition is administered in a dose offrom about 0.01 pmol/kg to 20 nmol/kg.
 4. The method of claim 1 whereinthe composition is administered as a single injection in a dose of fromabout 0.005 nmol/kg to 20 nmol/kg.
 5. The method of claim 1 wherein thecomposition is administered concurrently with glucose.
 6. The method ofclaim 1 wherein the composition is administered concurrently withpotassium.
 7. The method of claim 1 wherein the composition isadministered concurrently with a free radical scavenger.
 8. The methodof claim 1 wherein the composition is administered within four hours ofan ischemic event.
 9. The method of claim 8 wherein the compositioncontinues to be administered following the ischemic event.
 10. Themethod of claim 1 wherein the composition is administered concurrentlyor as soon as possible following therapies that reestablish flow in anartery that has been obstructed.
 11. The method of claim 1 wherein thecomposition is administered following a cardiac intervention selectedfrom the group consisting of angioplasty, coronary bypass grafting, andplacement of an intracoronary stent.
 12. The method of claim 11 whereinthe composition continues to be administered following the intervention.13. The method of claim 1 wherein the composition is administered totreat ventricular arrhythmias.
 14. The method of claim 13 wherein theventricular arrhythmia is caused by a condition selected from the groupconsisting of cardiac ischemia, cardiac ischemia-reperfusion, andcongestive heart failure.